12 QUANTIFICATION OF REACTIVE OXYGEN SPECIES IN BOVINE SEMEN WITH DIFFERENT SEMINAL QUALITY AND WITH EXTENDER CONTAINING ANTIOXIDANT EVALUATED BY ARTIFICIAL INSEMINATION

Abstract

The aim this study was to evaluate the effects on artificial insemination (AI) of minimizing oxidative stress by adding an antioxidant into the sperm microenvironment. For that purpose, semen of bulls with different seminal quality was evaluated in nature and after addition of extender with and without antioxidant, followed by freezing and thawing, with respect to thiobarbituric acid reactive substances (TBARS) concentration, total antioxidant capacity (TAC), and AI success. TBARS is an indicator of lipid peroxidation and TAC measures free radicals. Twelve ejaculates from 5 bulls were treated with Tris egg yolk extender (CE) and in half of each ejaculate the extender was supplemented with 200 μL of Trolox (AE). Three of these 5 bulls had high-quality semen (mean 11.9 ± 5.9 total defect, 7.7 ± 3.1 major defect, and 4.2 ± 3.9 minor defect) and 2 animals had poor-quality sperm (mean 31.9 ± 9.7 total defect, 26.3 ± 8.5 major defect, and 5.6 ± 5.0 minor defect) (P < 0.05). For AI, 300 Nelore heifers were divided in 10 groups and the females in each group were inseminated with semen of the same bull and extender treatment. To evaluate oxidative damage, TBARS concentration was measured to indirectly determine malondialdehyde (lipid-peroxidation metabolite) concentration, whereas TAC was measured using myoglobin, as a source of radicals, which interact with a chromogen 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), whose cation radical is spectroscopically detectable. The latency phase (lag) in the accumulation of ABTS cation is proportional to the antioxidant concentration. The pregnancy rates 60 days after AI were analyzed by chi-square test, and data of free radical production were evaluated by ANOVA, followed by the Tukey test with significance level of 5%. Reactive oxygen species (ROS) production in nature semen (0.351 nmol of TBARS in 50 × 10⁸ spermatozoa) was lower (P < 0.05) when compared to diluted semen (0.367 nmol of TBARS in 50 × 10⁸ spermatozoa). No difference (P > 0.05) between extenders was observed after thawing. The bulls with poor-quality semen presented total ROS production (0.967) superior (P < 0.05) to that of bulls with good-quality semen (0.753). Although the use of antioxidant in the extender did not decrease ROS and TAC production (P > 0.05), pregnancy rates after AI in bulls with poor quality semen with AE (60.0%) was superior (P < 0.06) to that in bulls with high-quality sperm with AE (56.6%). In conclusion, bulls with poor-quality semen present higher ROS and TAC production, and the use of antioxidant in the extender for cryopreservation with poor-quality bovine semen, in spite of not decreasing ROS and TAC production, improves pregnancy rates.

Financial support: FAPESP.